As one separation system of liquid chromatography, a reversed-phase liquid chromatography is widely known, where the separation is performed based on the strength of retention differing due to difference in the hydrophobicity between the packing material and the substance to be separated.
Conventionally, a packing material obtained by introducing an octadecyl group into a silanol group of silica gel using a silane coupling agent having an octadecyl group (this packing material is hereinafter referred to as an “ODS”) has been widely used as a packing material for reversed-phase liquid chromatography. This is because a fine particle having high mechanical strength can be produced relatively with ease.
However, silica gel is low in the alkali resistance and therefore, the usable eluent is limited to an eluent having a pH of 2 to 8. Furthermore, since it is actually impossible to chemically bond a coupling agent to all silanol groups, there arises a serious problem that a basic compound such as amine is readily adsorbed to an unreacted silanol group.
In recent years, various polymer-type packing materials for reversed-phase liquid chromatography have been developed so as to improve the handleability under alkali conditions, and some of them are commercially available as a column for reversed-phase liquid chromatography. Examples thereof include the followings:
(1) styrene-divinylbenzene copolymer particle ((see, Analytical Chemistry, Vol. 45, page 1383 (1973), commercially available product: Shodex (registered trademark of Showa Denko K.K.) RSpak RP18-413 produced by Showa Denko K.K.);
(2) methacrylate-type crosslinked polymer particle (commercially available product: Shodex (registered trademark of Showa Denko K.K.) RSpak DE-413 produced by Showa Denko K.K.);
(3) polyvinyl alcohol-type crosslinked polymer particle having chemically bonded thereto a long-chain acyl group (commercially available product: Shodex (registered trademark of Showa Denko K.K.) Asahipak (registered trademark of Showa Denko K.K.) ODP-40 4D produced by Showa Denko K.K.);
(4) methacrylic acid ester-type copolymer particle having a long-chain alkyl group (see, JP-A-2000-9707 (the term “JP-A” as used herein means an “unexamined published Japanese patent application”));
(5) copolymer particle of a glycidyl methacrylate and a (meth)acrylic acid ester of polyhydric alcohol, to which a long-chain alkyl group is chemically bonded (see, JP-A-61-272654); and
(6) hydroxyl group-containing methacrylate-type crosslinked polymer particle having chemically bonded thereto a long-chain acyl group (see, JP-A-4-58154).
The columns for reversed-phase liquid chromatography, packed with these polymer-type particles, have some problems which are not negligible while such a column is advantageous in that the usable pH range is broader than that of the ODS column.
However, the columns packed with particulate polymers of (1) to (5) are not suitable for the separation and analysis of natural products or medical preparations having a polycyclic aromatic site, because the chromatogram of polycyclic aromatic compound is broad.
In the column packed with the particulate polymer of (1), the particulate polymer itself heavily swells or shrinks depending on the solvent and it is difficult to satisfactorily perform the separation and analysis by varying the eluent in the column.
The columns packed with the particulate polymers of (2) to (6) have a problem in that when the column is used at a pH of 2 or less or a pH of 11 or more for a long period of time, since the ester bond contained in its structure has a low resistance to acid/alkali, the column efficiency seriously decreases, the peak shape of a basic substance such as amine changes for the worse and the measurement cannot be continuously performed.
As a method for overcoming such a problem, it would be effective to produce an ether bond which has a high resistance to acid/alkali by reacting an epoxy compound with a particulate polymer having a hydroxyl group in introducing a functional group. As examples of the method, (A) a method of performing the reaction in water containing a base such as sodium hydroxide or in a polar organic solvent such as dimethylformamide, and (B) a method of performing the reaction in an ether-type solvent such as dioxane in the presence of a Lewis acid such as boron trifluoride diethyl ether complex (see, for example, JP-A-61-272654 mentioned in above (5)) are known. However, these methods have a problem in that the reaction takes a long time and since an epoxy compound is used in an excess amount, the after-treatment such as filtration is very cumbersome. Moreover, if the substrate gel is polyester-type gel as in the case where the above-mentioned material (5) is used, when the reaction of (A) or (B) is performed, the ester bond of the particulate polymer itself is cut by the hydrolysis and a carboxyl group comes out because the substrate gel is exposed to an acid or an alkali for a long period of time. The particulate polymer where a carboxyl group is present adsorbs a basic substance such as amine and therefore, is not suitable as a packing material for reversed-phase liquid chromatography.
A packing material capable of overcoming the above-described problems and a production process therefor are not known and establishment of these is urgently demanded.